PROJECT SUMMARY Human type 1 diabetes (T1D) is characterized by the immune-mediated destruction of insulin-producing pancreatic beta cells. CD8 T cells are the most common [immune] cell found in insulitic lesions and are the principal T cell type implicated in beta cell destruction. Studies performed within this project have already revealed significant [and novel] findings including: 1) the first identification of auto-reactive CD8 T cells in the islets of patients with T1D using a specially [designed method of] tetramer staining and 2) [the first description of high] numbers of CD8 T cells infiltrating the exocrine pancreas in T1D patients compared with non-diabetic individuals. Based on these findings, we anticipate that only a proportion of the cells that infiltrate the pancreas are indeed auto-reactive. Thus, the pool of `bystander' CD8 T cells that recognize other, for example viral, antigens might significantly contribute to the inflammatory environment of the organ. At present, the overall specificities and frequencies of CD8 T cells in the pancreas and the cause for their entry and activation are not fully understood. Possible targets are known autoantigens derived from beta cells such as insulin, IGRP, IA-2 and GAD (see Table 2 for abbreviations) and cellular matrix proteins, which could become presented [and modified] when beta cells are destroyed, but viral proteins, for example enteroviral determinants are also possible candidates. The overall objective of this renewal application is therefore to compare the specificity, phenotype and function of CD8 T lymphocytes from human islets with those found in exocrine tissue, and to assess whether their presence correlates with islet-specific pathology (e.g. viral infections and their detectable footprints). We will study patients with recent-onset and longstanding T1D in order to build a road map of specificities and to further understand how the relationship between the CD8 T cell infiltrate and the disease course might evolve [both quantitatively and qualitatively over time. A unique strength is not only the access to very unique organ repositories but also the newly established collaboration with Dr. Alessandro Sette and Dr. Bjoern Peters' laboratories, which maintain the Immune Epitope Database (IEDB) at La Jolla institute and are part of an epitope discovery initiative led by Novo Nordisk. Using the information contained in the IEDB and in vitro assays, neo-epitopes to modified autoantigens and new viral CD8 T cell epitopes restricted to HLA-A2 will be mapped and the information shared with our laboratory. Overall, our findings should give us a better understanding of how and why T1D develops and thus help ultimately with the development of new therapeutic options.] Our first goal is to systematically and quantitatively detect autoreactive CD8 T cells within human islets and exocrine pancreas and to correlate the number and activation status of these cells with the local histopathology of the organ. In situ tetramer staining of freshly frozen human pancreata available from the Network for Pancreatic Organ Donors with Diabetes (nPOD), the Diabetes Biobank Brussels (DBB) and the Biobank at Oslo University Hospital will be performed. [The discovery of new post-translationally-modified epitopes using the information contained in the IEDB and the expertise at La Jolla Institute in conjunction with our experiments in situ will provide, for the first time,] information on the precise tissue distribution of antigen- specific CD8 T cells in the pancreas. In addition, we will further characterize their phenotype and function. This will allow for the identification of key antigens with significant polarization towards the islets, which are more likely to be involved in the pathogenesis of disease. It will also help us to understand why exocrine pancreas inflammation is often present in T1D. Our second goal is to search for virus-specific T cells within islets and exocrine tissue. [This will help to ascertain whether one virus might trigger the disease (hit-and-run event) or whether numerous viral attacks are required (multiple hits) or several viral strains of the same genus or even different viruses that trigger common pathways might play a role in the pathogenesis of the disease (multiple viruses)]. Pancreas tissue sections from donors with recent-onset and long duration of T1D will therefore be probed with virus-specific tetramers, and we will detect any viral proteins and nucleic acids in these samples in collaboration with the nPOD-Virology group in order to investigate the manner of viral infection (hit-and-run; multiple hits or multiple viruses). We will initially focus our attention on enteroviruses (EV), as only the association with group B coxsackieviruses has thus far been sufficiently documented. [For this purpose, we will aim to identify new EV epitopes through our collaboration with Dr. Sette and Dr. Peters' laboratories and the use of the IEDB as well as their expertise on the discovery and validation of cytotoxic and T cell epitopes presented by HLA Class I molecules]. The frequency of EV-specific cells will be compared to that of other common viruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV). We will also investigate the possible presence of herpesvirus-6 (HHV6) and HHV6-specific CD8 T cells, because recent evidence suggests that this virus might be present in the pancreas of T1D donors. In addition, we will correlate our findings with the local histopathology of the organ (MHC-I expression, interferon signature, CD8 T cell phenotype, cytokine production and presence/absence of viral protein or genome).